Metabolism of nitromiphene (CI 628) in the immature female rat: role of gastrointestinal microflora in the biotransformation of a triarylethylene antiestrogen.

Nitromiphene (NIT; CI 628) is a triarylethylene antiestrogen shown to be effective in treatment of experimental breast cancer. We have studied the fate of NIT in the immature female rat, the animal model in which most of the biochemical studies of NIT have been carried out. NIT was eliminated mainly via the feces after i.p. administration, primarily as metabolites. One of these, a diphenylmethane derivative, p-[2-(N-pyrrolidinyl)ethoxy]-p'-methoxybenzophenone (PMB), was also eliminated in urine as such and as its O-demethyl and keto-reduced metabolites. In uterine and liver tissue, unchanged NIT was accompanied by demethyl NIT (CI 628M), PMB, and a diarylacetophenone derivative, p-[2-(N-pyrrolidinyl)ethoxy-p'-hydroxybenzhydryl phenyl ketone (demethyl KET). In vitro studies showed that O-demethyl NIT was produced in the presence of liver enzymes and that PMB and demethyl KET were produced in the presence of intestinal bacteria. These results suggested that PMB and demethyl KET accumulate in uterine and liver tissue due to reabsorption from the intestine after having been produced there from NIT and demethyl NIT, respectively. The effects of antiestrogens and their metabolites may be due in part to interaction with antiestrogen binding sites. Both demethyl KET and PMB had good affinity for such sites. Thus, these enteric bacterial metabolites not only have the ability to accumulate in vivo, but could, together with demethyl NIT, contribute to the antiestrogenic effects observed with NIT.

Biotransformation of the antiestrogen clomiphene to chemically reactive metabolites in the immature female rat.

Novel metabolites of clomiphene (CLO), an antiestrogen effective in experimental breast cancer models, were characterized in studies using immature female rats. After i.p. administration, CLO was eliminated unchanged in feces and as two components which were chromatographically identical to synthetic CLO analogues bearing a m-methoxy-p-hydroxyphenyl (guaiacol) moiety in place of one or the other of its phenyl rings. These components were also found in liver tissue. In the presence of liver microsomal homogenate, CLO underwent p-hydroxylation of either of its phenyl rings, affording 4-hydroxy-CLO and 4'-hydroxy-CLO. These in turn underwent liver microsomal mediated conversion to the respective guaiacol metabolites. 4'-Hydroxy-CLO and its 3'-methoxy analogue, but not positional isomers of these, had arylating ability as shown by chemical and spectral studies, apparently due to spontaneous conversion to electrophilic allene-quinones. Reproductive tract abnormalities produced in neonatal rats by CLO were suggested not to be mediated via such metabolites, since similar such effects were caused by 4'-fluoro-CLO. However, the latent arylating potential of the 4'-hydroxylated metabolites of CLO suggests that these compounds may be useful in biochemical studies of breast cancer cell growth inhibition.

Specific bone-protective effects of metabolites/derivatives of tamoxifen and clomiphene in ovariectomized rats.

In the ovariectomized (ovx) rat, the nonsteroidal antiestrogens, clomiphene (CLO) and tamoxifen (TAM), at dose levels that prevent development of osteopenia to a degree approaching that of 17beta-estradiol are, in contrast to 17beta-estradiol, only weakly uterotrophic. Metabolites of CLO and TAM might contribute differentially to these effects. Thus, we have evaluated bone protective and uterine effects in ovx rats of two such metabolites: 4-hydroxy CLO, produced by p-hydroxylation of CLO; and 4HTA, produced from TAM by stepwise replacement of its dimethylaminoethyl side chain with an acetic acid moiety, accompanied by p-hydroxylation. Also reported are effects of D4HTA, the dihydrodesethyl derivative of 4HTA previously characterized as a full estrogen mimetic in vitro. Administration of 4-hydroxy CLO (2.5 mg/kg subcutaneously) 5 days/week for 5 weeks to 3-month-old ovx rats resulted in complete prevention of bone loss and suppression of bone turnover to levels comparable to those of intact controls and to those of ovx animals similarly receiving 17beta-estradiol (10 microg/kg). However, uterine weight in animals receiving 4-hydroxy CLO was 64% less than that in 17beta-estradiol-treated animals. Although 4HTA (3.7 mg/kg s.c.) had a modest uterotrophic effect, it did not prevent bone loss associated with ovariectomy. In contrast, D4HTA (3.6 mg/kg s.c.) partially reduced bone turnover indicators and cancellous bone loss in a manner similar in many ways to that observed in TAM-treated ovx animals, but it had no uterotrophic effect. These results suggest that, although 4HTA does not contribute to the bone-protective effect of TAM, 4-hydroxy CLO might augment that of CLO.

7-Aza analogs of the analgetic agent azabicyclane. Synthesis and pharmacologic analysis.

Three 3,7-diazabicyclo[3.3.1]nonane derivatives (4) with a structural similarity to the analgetic agent azabicyclane (1) were prepared. The amino alcohol 4a was found to prefer a conformation wherein the six-membered ring to which the hydroxyl group is syn is in the boat form. These three compounds had increased basicity in comparison with 1 due to various forces stabilizing their monocationic states. Compounds 4a-c did not show analgetic activity at the dose levels tested.

Analogues of sparteine. 5. Antiarrhythmic activity of selected N,N'-disubstituted bispidines.

A series of seven N,N'-disubstituted bispidines, structurally analogous to the inner (B and C) rings of sparteine (1) and encompassing a range of lipophilicity in which 1 was centered, has been compared to 1 in regard to antiarrhythmic potency and acute toxicity. Several of the bispidines were of comparable potency, and all but one were somewhat less toxic than 1. The ability of the mononitrate salts of 1 and bispidines 6 and 7 to bind calcium and magnesium cations in Me2SO-d6 solvent has been evaluated by proton magnetic resonance analysis. No binding could be demonstrated under these conditions, which suggested that pharmacologic effects of these compounds may be due to properties other than direct binding of these cations.

Antiarrhythmic activity of some N-alkylbispidinebenzamides.

A series of aromatic ring substituted bispidinebenzamides, 2--10, was prepared by condensation of N-methyl- or N-n-butylbispidine with the appropriate acid chlorides. These compounds were initially evaluated in mice for acute toxicity and for their ability to protect against chloroform-induced ventricular fibrillation. All compounds had significant activity, which was optimized in 2, 3, and 5. These last compounds had potencies and LD50/ED50 ratios comparable to those of a standard antiarrhythmic, disopyramide. However, their potencies in increasing the effective refactory period in isolated rabbit atria were considerably less than that of disopyramide.

Estrogenic and antiestrogenic activity of monophenolic analogues of tamoxifen, (Z)-2-[p-(1,2-diphenyl-1-butenyl)phenoxy]-N, N-dimethylethylamine.

Five hydroxylated analogues of tamoxifen [1, (Z)-2-[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine] and its geometric isomer were prepared by reaction of protected hydroxy-alpha-ethyldeoxybenzoins with 4-[2-dimethylamino) ethoxy]phenylmagnesium bromide, followed by acid-catalyzed dehydration-deprotection and chromatographic separation of isomer mixtures. Estrogen receptor binding affinity and estrogenic and antiestrogenic activity of each of the compounds were determined in the rat, in comparison with 4-hydroxytamoxifen (2). The new compounds had a wide range of receptor binding affinities, with that of 3-hydroxytamoxifen (6c), the most strongly bound, approaching that of estradiol. The trans isomers 6a,b were more strongly bound than were the cis isomers 7a,b. Antiestrogenic activity was seen in all compounds except 7b. This was also true for estrogenic activity, except that in 6c this activity was also substantially reduced. Maximal antiestrogenic effectiveness of 6c occurred at a 10-fold greater daily dose (50 micrograms/rat) than that required for maximal effect of 2.

Carboxylic acid analogues of tamoxifen: (Z)-2-[p-(1, 2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine. Estrogen receptor affinity and estrogen antagonist effects in MCF-7 cells.

The triarylethylene estrogen mimetic (E, Z)-4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxyacetic acid (4) represents a novel class of estrogen receptor (ER) ligands which, like tamoxifen (1), can elicit estrogen agonist and antagonist effects, in turn, in nonreproductive and reproductive tissues. Analogues of 4, incorporating structural features shown previously in triarylethylenes to improve ER affinity and estrogen antagonist properties, were prepared with the ultimate aim of identifying substances with improved estrogenicity exclusive of reproductive tissues. Thus, the side chain of 4 was elongated to give oxybutyric acid 13, which was further altered by (a) repositioning of its p-hydroxyl to the neighboring m-position (12) and (b) ethylenic bond reduction (14). Also, the p-hydroxyl group and oxyacetic acid groups of 4 were, in turn, shifted to the neighboring m-positions, affording 8 and 9. Oxybutyric acid analogue 13 had about 2 times the affinity for human ERalpha as 4, and its antiproliferative effect in MCF-7 cells was greater than that of 1. Dihydro analogue 14, which was conformationally similar to cis-13, had very low ER affinity and antiestrogenicity, and m-hydroxy analogue 12 also had reduced ER affinity and potency, but its MCF-7 cell antiproliferative efficacy was retained. Modest ER affinity and antiproliferative potency were seen with 8, in which phenolic and phenyl rings were trans to one another, but 9, in which these rings were cis, was inactive. Our findings indicate that two-carbon side-chain elongation and/or m-positioning of the hydroxyl group in 4 affords analogues with dominant estrogen antagonist effects in MCF-7 cells.

Synthesis and estrogen receptor selectivity of 1,1-bis(4-hydroxyphenyl)-2-(p-halophenyl)ethylenes.

A series of triarylethylenes (1a-e) were synthesized and evaluated for their ability to compete with [3H]estradiol for high-affinity estrogen receptors (ER) in immature rat uterine cytosol. All compounds showed affinity comparable to that of estradiol, with 1c having the highest affinity and the lowest calculated nonspecific binding of the para-halogenated members. Compound 1a had a higher affinity than did its chlorovinyl counterpart 1b, indicating that a vinyl hydrogen was suitable for high ER affinity in this series. Compound 1c was labeled with 3H ortho to one or both of its hydroxyls. Its ratio of specific to nonspecific binding in rat uterine cytosol, 3.2, was 140% of that of a related triarylethylene, 4-hydroxytamoxifen, and was 24% that of estradiol. Administration of [3H]-1c to immature female rats resulted in accumulation of 3H in uterine tissue which was decreased 39% when [3H]-1c was coadministered with estradiol. The major site of accumulation 1, 4, and 8 h after administration was in the intestinal tract. Chromatographic analysis showed that levels of 1c were less than those of 1c glucuronide in blood plasma, liver, and intestinal contents of rats 1 h after administration of 1c. Uterine 3H was comprised of 85% of 1c and 11% of 1c glucuronide. These results indicate that 1c undergoes ER-mediated uptake in the immature female rat, but selectivity is reduced due to nonspecific accumulation of free and conjugated 1c in uterine tissue.

Estrogen receptor binding and estrogenic/antiestrogenic effects of two new metabolites of nitromiphene, 2-[p-[2-nitro-1-(4-methoxyphenyl)-2-phenylvinyl]phenoxy]-N-ethylpyrrolidine.

Reduction of the triarylethylene antiestrogen 2-[p-[2-nitro-1-(4-methoxphenyl)-2-phenylvinyl]phenoxy]-N-ethylpyrrolidine (1) with sodium borohydride-stannous chloride afforded 2-(p-methoxyphenyl)-p'-(2-pyrrolidin-1-ylethoxy)deoxybenzoin (2). Incubation of 1 with rat cecal content suspension under aerobic or anaerobic conditions also resulted in the generation of 2. The lactam analogue of 1 (6) was prepared by condensation of 4-(2-bromoethoxy)-4'-methoxybenzophenone (3) with benzylmagnesium chloride, followed by dehydration, amidation with 2-pyrrolidinone, and nitration. A metabolite with chromatographic and spectral properties identical with those of 6 was found in extracts from incubation mixtures of 1 with phenobarbital-induced rat liver 9000g supernatant. Compound 2 did not exhibit appreciable binding to the rat uterine cytosol estrogen receptor at concentrations of up to 1 X 10(-6) M and had no estrogenic or antiestrogenic activity in the 3-day rat uterotropic assay. By contrast, 6 had estrogen receptor affinity somewhat greater than that of 1 and slightly greater estrogenic activity accompanied by reduced antiestrogenic activity in comparison with those of 1.

Phenolic metabolites of clomiphene: [(E,Z)-2-[4-(1,2-diphenyl-2-chlorovinyl)phenoxy]ethyl]diethylamine. Preparation, electrophilicity, and effects in MCF 7 breast cancer cells.

The triarylethylene antiestrogen clomiphene was previously shown to undergo biotransformation to an active metabolite, 4-hydroxyclomiphene, and to 3-methoxy-4-hydroxyclomiphene plus the respective regioisomers of these, 4 and 5. We now report the synthesis and further chemical and biochemical studies on 3-5. Coupling of 4-[2-(diethylamino)ethoxy]benzophenone with either 4-(benzyloxy)benzaldehyde or its 3-methoxy analogue 11b in the presence of titanium, followed by chlorination and deprotection of the intermediate triarylethylenes, gave 4 and 5, respectively. Condensation of benzylmagnesium chloride with the (2-methoxyethoxy)methyl (MEM) ether of 4-[2-(diethylamino)ethoxy]-3'-methoxy-4'-hydroxybenzophenone, followed by mild acid treatment, afforded deschloro 3 due to facile MEM ether hydrolysis. Acetylation of this, followed by chlorination and deacetylation, gave 3. Compounds 4 and 5 reacted readily with nucleophiles. In particular, 2-mercaptoethanol reacted with 4 to afford deschloro vinyl thioether 13 as suggested by NMR spectral studies, a result that implicated allene-quinone 14 as the electrophilic species produced in solution from 4. Antiestrogen binding sites and estrogen receptors from MCF 7 human breast cancer cells interacted with 3 and 5 with affinities comparable to those of tamoxifen and 1, respectively; 5 was shown not to bind irreversibly with these sites. Inhibition of MCF 7 cell proliferation by 3-5 at 5 microM concentrations (76%, 57%, and 49%, respectively, relative to drug-free controls) compared favorably to that observed with 5 microM 1 (80%). These results suggest that 3-5 as well as 2 may contribute to the antiestrogenic effects of 1.

Estrogenic triarylethylene acetic acids: effect of structural variation on estrogen receptor affinity and estrogenic potency and efficacy in MCF-7 cells.

Triarylethylenecarboxylic acids exemplified by (E,Z)-2-{4-[1-(p-hydroxyphenyl)-2-phenyl]-1-butenyl}phenoxyacetic acid (8) are a new class of estrogen receptor (ER) ligands capable of tissue selective estrogen agonist and antagonist effects. We report the syntheses of 8 and of analogues incorporating structural features known or anticipated to facilitate ER affinity in triarylethylenes. These studies revealed that the p-hydroxyphenyl moiety, ethylenic bond, and ether oxygen of 8 were all critical for high ER affinity. Although a 1,1-bisphenolic analogue bearing the p-(oxyacetic acid) moiety on its 2-phenyl ring, 12, had low ER affinity, it exhibited estrogenic potency approaching that of 8 in MCF-7 cells. Unlike 8 which was a partial agonist with weak antagonist potency, 12 was a full agonist. A similar profile of potency/efficacy in MCF-7 cells was seen in 9, an ethylenic bond saturated analogue of 8. Growth-promoting effects of 8, 9, and 12 were fully antagonized by the antiestrogen tamoxifen, suggesting that such effects were mediated solely via ER. Thus, our studies in MCF-7 cells have confirmed the estrogenicity of 8 and have enabled identification of two analogues with favorable estrogenic potency and full estrogen efficacy. On this basis, these three (triarylethylene)acetic acids have been selected for more intensive animal studies of their extrareproductive tract estrogenic effects.

Substituted-vinyl hydroxytriarylethylenes, 1-[4-[2-(diethylamino) ethoxy]phenyl]-1-(4-hydroxyphenyl)-2-phenylethylenes: synthesis and effects on MCF 7 breast cancer cell proliferation.

A series of triarylethylene compounds related to 4-hydroxyclomiphene (2) in which the vinyl Cl substituent was replaced by ethyl (5), Br (6), H (7), CN (8), or NO2 (9) substituents were synthesized to facilitate studies of the molecular actions of synthetic nonsteroidal antiestrogens. The relative binding affinities of these compounds for the estrogen receptor (ER) and the antiestrogen binding site (AEBS) in MCF 7 human mammary carcinoma cells were measured and correlated with the effects of these drugs on cell proliferation kinetics. Affinities for ER and AEBS were highly correlated, illustrating that vinyl substituents influence binding to ER and AEBS in a parallel manner. All compounds except 7 had biphasic effects on cell proliferation kinetics, indicating the presence of at least two distinct mechanisms by which hydroxytriarylethylenes inhibit breast cancer cell proliferation. In the concentration range 10(-10)-10(-8) M, cell proliferation was inhibited by 60-70%, these effects were estrogen-reversible, and the degree of growth inhibition was in the order Cl greater than Et greater than Br greater than NO2 greater than CN greater than H, which paralleled the order of affinities for ER. There was no further inhibition of cell growth between 10(-8) and 10(-6) M, but at concentrations greater than 10(-6) M there was a further dose-dependent decrease in cell growth mediated by mechanisms yet to be defined but apparently distinct from ER-mediated events. In both concentration ranges, growth inhibition was accompanied by accumulation of cells in the G1 phase of the cell cycle. These data, obtained with a novel series of hydroxytriarylethylenes, have enabled clear definition of two distinct mechanisms of growth inhibition by triarylethylene antiestrogens. They also indicate that among the vinyl substitutions examined to date the Cl substituent yields the most active molecule both in terms of affinity for ER and AEBS and potency as a growth inhibitory agent.

Antiestrogens. 3. Estrogen receptor affinities and antiproliferative effects in MCF-7 cells of phenolic analogues of trioxifene, [3,4-dihydro-2-(4- methoxyphenyl)-1-naphthalenyl][4-[2-(1-pyrrolidinyl)ethoxy]- phenyl]methanone.

Benzothiophenes 3 and 4, derived from the acrylophenone antiestrogen trioxifene (2), are characterized by high estrogen receptor (ER) affinity and low residual estrogenicity compared to tamoxifen (1a). In order to characterize further the growth suppression mechanism for these structural types we have prepared structural variants of 2 bearing hydroxy groups positioned to maximize ER affinity. Thus, dihydronaphthalenes 5 and 6 and benzofluorenes 7 and 8 were prepared and studied in MCF-7 human breast cancer cells, in comparison with 3 and 4. All compounds were powerful suppressants of cell growth, with 50% inhibition ranging from 4.5 to 160 nM. Greatest potency was seen with diphenols 6 and 8. These compounds had intracellular ER affinities ranging from 0.2 to 4.1% of that of estradiol, suggestive of a potential for partial agonist effects. Simultaneous exposure of cells to 0.1 microM concentrations of estradiol and 3 or 4 did not affect the degree of growth inhibition seen with 0.1 microM 3 or 4 alone. Partial reversal of inhibition occurred when 0.1 microM 5-8 were each accompanied by 0.1 microM estradiol. Under these conditions complete reversal of growth inhibition has been found with 1a, 1b, and other triarylethylenes. Calmodulin, a putative target for triarylethylenes, and which is antagonized by 1a, was shown to interact weakly with 7 and 8 and not at all with 3-6. These results suggest that MCF-7 cell growth suppression by 3-8 may be due to interaction with unidentified receptors besides ER and extend earlier findings indicating that events occurring after interaction of these compounds with ER differ from those of triarylethylene antiestrogens.

Resolution, absolute configuration, and antiarrhythmic properties of the enantiomers of disopyramide, 4-(diisopropylamino)-2-(2-pyridyl)-2-phenylbutyramide.

Disopyramide was resolved by fractional crystallization of its diastereomeric bitartrate salts from methanol-acetone. Diastereomeric sulfonamides prepared from (+)-camphor-10-sulfonyl chloride and the primary amines obtained by LiAlH4 reduction of the resolved bases were separable by high-performance LC and were used to show that within experimental error resolution of disopyramide was complete. The absolute configuration was determined by X-ray crystallography. (+)-[(2R)-(-)-4-(Diisopropylamino)-2-(2-pyridyl)-2-phenylbutyramide (+)-(2R,3R)-bitartrate] crystallizes in space group P212121: a = 14.789 (4), b = 18.151 (4), c = 9.878 (2) A; Z = 4: Dx = 1.225, Dm (flotation C6H6/CCl4) = 1.226 g cm-3. The structure was solved by direct methods. The enantiomeric bitartrates were tested for antiarrhythmic properties. The enantiomeric bitartrate salts were equally effective in prolonging the effective refractory period (ERP) of rabbit ventricle. At 3 x 10(-6) M, the (-)-bitartrate [from (2S)-(+)-disopyramide] increased the ERP 21.8 +/- 6.3 ms and the (+)-bitartrate [from (2R)-(-)-disopyramide] increased the ERP 25.8 +/- 3.6 ms. At 1.5 x 10(-5) M no significant difference was noted, as the increases in the ERP were 41.2 +/- 8.9 and 50.5 +/- 6.3 ms for the (-)- and %+)-bitartrate, respectively.

Synthesis, conformational analysis, and antiarrhythmic properties of 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonan-9-one, 7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane hydroperchlorate, and 7-benzyl-9-phenyl-3-thia-7-azabicyclo[3.3.1]nonan-9-ol hydroperchlorate and derivatives: single-crystal X-ray diffraction analysis and evidence for chair-chair and chair-boat conformers in the solid state.

The synthesis of the title ketone has been completed via a type of Mannich reaction starting from 4- thianone . An X-ray diffraction analysis has revealed that the solid system is a chair-boat conformer with the sulfur atom in the boat portion of the bicyclic ring compound. Wolff- Kishner reduction of the ketone group gave 7-benzyl-3-thia-7-azabicyclo [3.3.1]nonane, which was isolated as the hydroperchlorate . However, X-ray diffraction analysis of the salt showed this solid to be a chair-chair conformer. Addition of phenylmagnesium bromide to the ketone gave a tertiary alcohol with the C-C6H5 bond being equatorial with respect to the thiane ring and axial with respect to the piperidine ring. The reaction of the Grignard reagent with the ketone to give this alcohol seems to be very stereospecific. An X-ray analysis of the hydroperchlorate of the alcohol confirmed the system to be a chair-chair form in the solid. The title compounds were screened for antiarrhythmic activity in anesthetized mongrel dogs in which myocardial infarctions had been created when the left anterior descending coronary artery was ligated. Vagal-induced slowing of the sinus mode firing rate was used to determine the underlying ventricular automaticity in the dogs, which averaged 164 +/- 27 beats/min. Ventricular pacing was initiated to rates between 240 and 390/min. This technique resulted in the induction of rapid and sustained ventricular tachycardia. At doses of 3 and 6 mg/kg of body weight, 7-benzyl-3-thia-7-azabicyclo [3.3.1]nonane hydroperchlorate in alcohol (the solution was administered intravenously) was able to suppress markedly the induced ventricular tachycardia in five of six dogs. The compound also caused a 10-15% increase in blood pressure within a few minutes. The antiarrhythmic properties of this compound and others of related structure are discussed, and some comparison is made with the action of lidocaine in similar dog preparations.

Metabolism of clomiphene in the rat. Estrogen receptor affinity and antiestrogenic activity of clomiphene metabolites.

Incubation of the nonsteroidal antiestrogen clomiphene with rat liver microsomes resulted in the formation of the 4-hydroxy-, N-desethyl-, and N-oxide metabolites, in qualitative contrast to results previously obtained analogously with rabbit microsomes, with which only the first two metabolites were detected. Metabolites were characterized by thin-layer chromatography in comparison with synthetic standards. They were similarly compared using low resolution electron ionization mass spectrometry, except for the N-oxide which was best characterized by fast atom bombardment mass spectrometry. Oral administration of clomiphene resulted in no detectable urinary elimination of the drug or its metabolites; 4-hydroxyclomiphene was the sole detectable elimination product in fecal extracts. The relative uterine cytosol estrogen receptor binding affinities, at 4 degrees, of 4-hydroxyclomiphene and the E-isomers of clomiphene, desethylclomiphene, and clomiphene N-oxide were, in turn, 331, 0.71, 0.62, and 0.88 (estradiol = 100). In the 3-day immature rat uterotropic assay, 4-hydroxyclomiphene had no significant uterotropic effect at doses up to 50 micrograms/day, but substantially inhibited that of estradiol (0.5 micrograms/day) at doses of 2 micrograms/day.

Estrogen receptor affinity and effects on MCF-7 cell growth of triarylethylene carboxylic acids related to tamoxifen.

The estrogen receptor binding, and growth suppressant and stimulating effects in MCF-7 human breast cancer cells, of four structural variants of the triarylethylene antiestrogen tamoxifen (1) were studied. In these analogs, the dialkylaminoethoxy side chain of 1 was replaced by carboxylic acid or oxyacetic acid substituents. The presence of a p-hydroxy group in the ring geminal to the one bearing the side chain resulted in ligands with estrogen receptor affinities greater than that of 1 but less than that of estradiol. Compared to 1, none of the test compounds were effective suppressants of cell growth. To the contrary, the phenolic oxyacetic acid analog effectively reversed the growth suppressive effect of 1. Also, it was as effective as estradiol, though less potent, in stimulating growth of cells grown in estrogen depleted medium, suggestive of full estrogen agonist activity. Its carboxylic acid counterpart had little or no effect on proliferation. Because the phenolic oxyacetic acid is a metabolite of 1 in animals, its estrogenicity may have therapeutic implications of concern, depending on the extent to which it is formed and distributed in tissues of patients receiving 1.

Estrogenic tamoxifen derivatives: categorization of intrinsic estrogenicity in MCF-7 cells.

Triarylethylenes bearing acetic acid side chains, exemplified by 4-[1-(p-hydroxyphenyl)-2-phenyl-1-butenyl]phenoxyacetic acid (4HTA), a derivative of tamoxifen (TAM), are of current interest as estrogen mimics lacking reproductive tract effects. Affinities for estrogen receptors (ER) and effects on cell growth kinetics of a diverse series of such compounds were compared with 4HTA, TAM, and with standard estrogens 17beta-estradiol (E2) and chlorotrianisene (CTA) in MCF-7 cells. These compounds exhibited concentration dependent cell growth stimulation comparable to that of CTA but less than that of E2. Growth stimulation of the more potent compounds was antagonized by TAM, signifying that effects were mediated via interaction with ER. At concentrations of 1 microM or higher, compounds with efficacies less than that of E2 were weak antagonists of estradiol-stimulated growth. Both intracellular ER affinities and growth rate stimulation potencies of the triarylethylene acetic acids and the standard ER ligands varied over a range of nearly three orders of magnitude. Analysis of growth stimulatory potency as a function of ER affinity revealed dual parallel correlations: the potency/ER affinity ratios of 4HTA and four of its analogues was about 100-fold less than those of the hydroxytriarylethane and bisphenolic analogs and the three standard ER ligands. These results suggested that ER liganded with the latter substances is more 'effective' at nuclear effector sites than is ER liganded with 4HTA and the other acidic triarylethylenes.

Formation of benzophenone and alpha, alpha-diarylacetophenone metabolites of the antiestrogen nitromiphene (CI628) in the presence of rat cecal contents.

Incubation of the nonsteroidal antiestrogen nitromiphene (CI628) with rat cecal content suspension under aerobic or anaerobic conditions resulted in extensive biotransformation, yielding three metabolites, as determined by thin-layer chromatography. These metabolites were not recovered from incubation mixtures containing previously frozen suspension, and recoveries were decreased (that of nitromiphene was increased) when incubations were carried out in the presence of 2mM EDTA. Spectral and chromatographic comparison of two of the purified metabolites resulted in their structural characterization, as p-[beta(N-pyrrolidinyl)ethoxy]p'-methoxybenzophenone, and a similarly substituted alpha, alpha-diphenyl-acetophenone. These metabolites are, in turn, due formally to ethylenic bond cleavage and nitro group reduction/hydrolysis of nitromiphene.